Through rational drug design, medicinal Chemists have synthesized many peptide mimetics with novel therapeutic potential. However, because of their low permeability through biological barriers (e.g., intestinal mucosa, brain endothelial cells) and their propensity to be cleared by the liver and eliminated in the bile, peptide mimetics have been difficult to develop as therapeutic agents. For example, peptide mimetics in general exhibit extremely low oral bloavailability which can be attributed to low permeability through the intestinal mucosal and/or rapid first pass clearance by the liver. In addition, even when peptide mimetics are administered via a parenteral route (e.g., intravenous), they are in general rapidly cleared by the liver and tend not to gain access to important target organs (e.g., brain). Therefore, the overall objectives of this research project are to elucidate what effects various bioisosters of the peptide bond nave on permeability through biological barriers like the intestinal mucosa and the blood brain barrier (BBB) and what effects these structural modifications have on first pass clearance by the liver and elimination into the bile. These objectives will be accomplished by synthesizing a series of di and tripeptides containing commonly used bioisosters of the peptide bond. We will then determine their overall permeability and their pathway(s) of permeation through the intestinal mucosa, using an in vitro cell culture model (Caco-2) and an in situ rat intestinal perfusion model, and through the BBB, using an in vitro cell culture model (cultured bovine brain microvessel endothelial cells) and an in situ rat brain perfusion model. Experiments will be designed to elucidate the contribution of both passive and carrier-mediated pathways (e.g., peptide transporter in the intestinal mucosa) of flux and to determine if their permeability is limited by their substrate properties for an apically polarized efflux mechanism (p-glycoprotein) present in the intestinal mucosa and the BBB. Experiments will also be conducted to determine the liver clearance properties of these peptide mimetics using an in situ perfused rat liver model and isolated hepatocytes. Experiments will be designed to elucidate the contributions of both passive and carrier-mediated Pathways (e.g., sodium taurocholate co-transporters). Based on the results of this project, knowledge about the "structure transport" relationships for commonly used peptide bioisosters will be forthcoming. This information should allow medicinal chemists to do both "rational drug delivery" (focused on transport across biological barriers) and "rational drug design" (focused on the pharmacological target) which will ultimately lead to the design of peptide mimetics that can be developed as clinical agents.